The methods of this invention relate to systems for genetic identification for disease states and other gene related afflictions. More particularly, the methods relate to systems for the detection of single nucleic acid polymorphisms in nucleic acid sequences for the identification of polymorphisms in viruses, and eukaryotic and prokaryotic genomes.
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein sequences. Many of these techniques and procedures form the basis of clinical diagnostic assays and tests. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and the separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugation, and electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility.
For example, the complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved. Broadly speaking, the complete process may be divided into a number of steps and sub-steps. In the case of genetic disease diagnosis, the first step involves obtaining the sample (e.g., saliva, blood or tissue). Depending on the type of sample, various pre-treatments would be carried out. The second step involves disrupting or lysing the cells which releases the crude DNA material along with other cellular constituents.
Generally, several sub-steps are necessary to remove cell debris and to further purify the DNA from the crude sample. At this point several options exist for further processing and analysis. One option involves denaturing the DNA and carrying out a direct hybridization analysis in one of many formats (dot blot, microbead, microplate, etc.). A second option, called Southern blot hybridization, involves cleaving the DNA with restriction enzymes, separating the DNA fragments on an electrophoretic gel, blotting the DNA to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the genomic DNA sample, and thereby helps to improve the hybridization specificity and sensitivity. Unfortunately, this procedure is long and arduous. A third option is to carry out an amplification procedure such as the polymerase chain reaction (PCR) or the strand displacement amplification (SDA) method. These procedures amplify (increase) the number of target DNA sequences relative to non-target sequences. Amplification of target DNA helps to overcome problems related to complexity and sensitivity in genomic DNA analysis. After these sample preparation and DNA processing steps, the actual hybridization reaction is performed. Finally, detection and data analysis convert the hybridization event into an analytical result.
Nucleic acid hybridization analysis generally involves the detection of a very small number of specific target nucleic acids (DNA or RNA) with an excess of probe DNA, among a relatively large amount of complex non-target nucleic acids. A reduction in the complexity of the nucleic acid in a sample is helpful to the detection of low copy numbers (i.e. 10,000 to 100,000) of nucleic acid targets. DNA complexity reduction is achieved to some degree by amplification of target nucleic acid sequences. (See, M. A. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990, Spargo et al., 1996, Molecular and Cellular Probes, in regard to SDA amplification). This is because amplification of target nucleic acids results in an enormous number of target nucleic acid sequences relative to non-target sequences thereby improving the subsequent target hybridization step.
The actual hybridization reaction represents one of the most important and central steps in the whole process. The hybridization step involves placing the prepared DNA sample in contact with a specific reporter probe at set optimal conditions for hybridization to occur between the target DNA sequence and probe.
Hybridization may be performed in any one of a number of formats. For example, multiple sample nucleic acid hybridization analysis has been conducted in a variety of filter and solid support formats (See G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985). One format, the so-called xe2x80x9cdot blotxe2x80x9d hybridization, involves the non-covalent attachment of target DNAs to a filter followed by the subsequent hybridization to a radioisotope labeled probe(s). xe2x80x9cDot blotxe2x80x9d hybridization gained wide-spread use over the past two decades during which time many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridizationxe2x80x94A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985). For example, the dot blot method has been developed for multiple analyses of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
New techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional xe2x80x9cdot blotxe2x80x9d and xe2x80x9csandwichxe2x80x9d hybridization systems.
The micro-formatted hybridization can be used to carry out xe2x80x9csequencing by hybridizationxe2x80x9d (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers (n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (see R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
There are two formats for carrying out SBH. The first format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence. The second format involves attaching the target sequence to a support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations.
Southern, (United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992), proposed using the first format to analyze or sequence DNA. Southern identified a known single point mutation using PCR amplified genomic DNA. Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH. However, Southern did not address how to achieve optimal stringency conditions for each oligonucleotide on an array.
Drmanac et al., (260 Science 1649-1652, 1993), used the second format to sequence several short (116 bp) DNA sequences. Target DNAs were attached to membrane supports (xe2x80x9cdot blotxe2x80x9d format). Each filter was sequentially hybridized with 272 labeled 10-mer and 11-mer oligonucleotides. Wide ranges of stringency conditions were used to achieve specific hybridization for each n-mer probe. Washing times varied from 5 minutes to overnight using temperatures from 0xc2x0 C. to 16xc2x0 C. Most probes required 3 hours of washing at 16xc2x0 C. The filters had to be exposed from 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced set of oligomer probes, and the use of the most stringent conditions available.
Currently, a variety of methods are available for detection and analysis of the hybridization events. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorimetrically, colorimetrically, or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or particle emission, information may be obtained about the hybridization events. Even when detection methods have very high intrinsic sensitivity, detection of hybridization events is difficult because of the background presence of non-specifically bound materials. Thus, detection of hybridization events is dependent upon how specific and sensitive hybridization can be made. Concerning genetic analysis, several methods have been developed that have attempted to increase specificity and sensitivity.
One form of genetic analysis is analysis centered on elucidation of single nucleic acid polymorphisms or (xe2x80x9cSNPsxe2x80x9d). Factors favoring the usage of SNPs are their high abundance in the human genome (especially compared to short tandem repeats, (STRs)), their frequent location within coding or regulatory regions of genes (which can affect protein structure or expression levels), and their stability when passed from one generation to the next (Landegren et al., Genome Research, Vol. 8, pp. 769-776, 1998).
A SNP is defined as any position in the genome that exists in two variants and the most common variant occurs less than 99% of the time. In order to use SNPs as widespread genetic markers, it is crucial to be able to genotype them easily, quickly, accurately, and cost-effectively. It is of great interest to type both large sets of SNPs in order to investigate complex disorders where many loci factor into one disease (Risch and Merikangas, Science, Vol. 273, pp. 1516-1517, 1996), as well as small subsets of SNPs previously demonstrated to be associated with known afflictions.
Numerous techniques are currently available for typing SNPs (for review, see Landegren et al., Genome Research, Vol. 8, pp. 769-776,1998), all of which require target amplification. They include direct sequencing (Carothers et al., BioTechniques, Vol. 7, pp. 494-499, 1989), single-strand conformation polymorphism (Orita et al., Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 2766-2770, 1989), allele-specific amplification (Newton et al., Nucleic Acids Research, Vol. 17, pp. 2503-2516, 1989), restriction digestion (Day and Humphries, Analytical Biochemistry, Vol. 222, pp. 389-395, 1994), and hybridization assays. In their most basic form, hybridization assays function by discriminating short oligonucleotide reporters against matched and mismatched targets. Due to difficulty in determining optimal denaturation conditions, many adaptations to the basic protocol have been developed. These include ligation chain reaction (Wu and Wallace, Gene, Vol. 76, pp. 245-254, 1989) and minisequencing (Syvxc3xa4nen et al., Genomics, Vol. 8, pp. 684-692, 1990). Other enhancements include the use of the 5xe2x80x2-nuclease activity of Taq DNA polymerase (Holland et al., Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 7276-7280, 1991), molecular beacons (Tyagi and Kramer, Nature Biotechnology, Vol. 14, pp.303-308, 1996), heat denaturation curves (Howell et al., Nature Biotechnology, Vol. 17, pp. 87-88, 1999) and DNA xe2x80x9cchipsxe2x80x9d (Wang et al., Science, Vol. 280, pp. 1077-1082, 1998). While each of these assays are functional, they are limited in their practical application in a clinical setting.
An additional phenomenon discovered to be useful in distinguishing SNPs is the nucleic acid interaction energies or base-stacking energies derived from the hybridization of multiple target specific probes to a single target. (see R. Ornstein et al., xe2x80x9cAn Optimized Potential Function for the Calculation of Nucleic Acid Interaction Energiesxe2x80x9d, in Biopolymers, Vol.17, 2341-2360 (1978); J. Norberg and L. Nilsson, Biophysical Journal, Vol. 74, pp. 394-402, (1998); and J. Pieters et al., Nucleic Acids Research, Vol.17, no. 12, pp. 4551-4565 (1989)). This base-stacking phenomenon is used in a unique format in the current invention to provide highly sensitive Tm differentials allowing the direct detection of SNPs in a nucleic acid sample.
Prior to the format of the current invention, other methods have been used to distinguish nucleic acid sequences in related organisms or to sequence DNA. For example, U.S. Pat. No. 5,030,557 by Hogan et al. disclosed that the secondary and tertiary structure of a single stranded target nucleic acid may be affected by binding xe2x80x9chelperxe2x80x9d oligonucleotides in addition to xe2x80x9cprobexe2x80x9d oligonucleotides causing a higher Tm to be exhibited between the probe and target nucleic acid. That application however was limited in its approach to using hybridization energies only for altering the secondary and tertiary structure of self-annealing RNA strands which if left unaltered would tend to prevent the probe from hybridizing to the target.
With regard to DNA sequencing, K. Khrapko et al., Federation of European Biochemical Societies Letters, Vol. 256, no. 1,2, pp. 118-122 (1989), for example, disclosed that continuous stacking hybridization resulted in duplex stabilization. Additionally, J. Kieleczawa et al., Science, Vol. 258, pp. 1787-1791 (1992), disclosed the use of contiguous strings of hexamers to prime DNA synthesis wherein the contiguous strings appeared to stabilize priming. Likewise, L. Kotler et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 4241-4245, (1993) disclosed sequence specificity in the priming of DNA sequencing reactions by use of hexamer and pentamer oligonucleotide modules. Further, S. Parinov et al., Nucleic Acids Research, Vol. 24, no. 15, pp. 2998-3004, (1996), disclosed the use of base-stacking oligomers for DNA sequencing in association with passive DNA sequencing microchips. Moreover, G. Yershov et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 4913-4918 (1996), disclosed the application of base-stacking energies in SBH on a passive microchip. In Yershov""s example, 10-mer DNA probes were anchored to the surface of the microchip and hybridized to target sequences in conjunction with additional short probes, the combination of which appeared to stabilize binding of the probes. In that format, short segments of nucleic acid sequence could be elucidated for DNA sequencing. Yershov further noted that in their system the destabilizing effect of mismatches was increased using shorter probes (e.g., 5-mers). Use of such short probes in DNA sequencing provided the ability to discern the presence of mismatches along the sequence being probed rather than just a single mismatch at one specified location of the probe/target hybridization complex. Use of longer probes (e.g., 8-mer, 10-mer, and 13-mer oligos) were less functional for such purposes.
An additional example of methodologies that have used base-stacking in the analysis of nucleic acids includes U.S. Pat. No. 5,770,365 by Lane et al., wherein is disclosed a method of capturing nucleic acid targets using a unimolecular capture probe having a single stranded loop and a double stranded region which acts in conjunction with a binding target to stabilize duplex formation by stacking energies.
Despite the knowledge of base-stacking phenomenon, applications as described above have not resulted in commercially acceptable methods or protocols for either DNA sequencing or the detection of SNPs for clinical purposes. We provide herein such a commercially useful method for making such distinctions in numerous genetic and medical applications by combining the use of base-stacking principles and electronically addressable microchip formats.
Methods are provided for the analysis and determination of SNPs in a genetic target. In one embodiment of the invention, SNPs in a target nucleic acid are determined using a single capture site on an electronically addressable microchip (e.g, an APEX type microchip). In this embodiment, both wild type and mutant alleles are distinguished, if present in a sample, at a single capture site by detecting the presence of hybridized allele-specific probes labeled with fluorophores sensitive to excitation at various wave lengths. In another embodiment, base-stacking energies of at least two oligonucleotides are used in conjunction with an APEX type bioelectronic microchip.
The electronically facilitated method using an APEX type microchip offers several advantages over passive-based hybridization assays when base-stacking is employed. First, electronic addressing under low salt conditions in the presence of stabilizer oligomer inhibits rehybridization of amplicon strands in situations where amplification of target nucleic acid is carried out. This obviates the need for asymmetric amplification or other more complex methods of strand separation. Electronically facilitated methods additionally allow multiple different amplicons to be addressed to discrete sites thereby greatly facilitating multiplexing of multiple patients or multiple amplicons on an open microchip.
In one embodiment of our system, the amplicons of the target nucleic acid may be anchored to an electronic microchip capture site (i.e. xe2x80x9camplicon downxe2x80x9d format) such that multiple amplicons may be placed at the same capture site. The amplicons may be anchored to the capture site on the microchip by attachment moieties located at the 5xe2x80x2 end of the amplicon. Such attachment moieties can be binding agents such a biotin incorporated into one of the amplification primers. The anchored nucleic acids may in turn be probed simultaneously or sequentially.
By way of example, in implementation of the amplicon down format, a target nucleic acid is first amplified, such as by PCR, SDA, NASBA, TMA, rolling circle, T7, T3, or SP6, each of which methods are well understood in the art, using at least one amplification primer oligomer that is labeled with a moiety useful for attaching the amplification product to a substrate surface. In one embodiment, a biotin moiety can be attached at the 5xe2x80x2 end of the primer. Following amplification, the labeled amplified dsDNA product may be denatured electronically or thermally and addressed to a specified capture site on the microchip surface, thereby making the amplicon behave as an anchored capture moiety. In a preferred embodiment, the complementary strand to the labeled amplification product (i.e., the non-labeled strand) is kept from reannealing to the labeled product by a xe2x80x9cstabilizerxe2x80x9d oligomer which is inputted into the process during electronic biasing of the labeled targeted amplicon to the capture site. The use of a xe2x80x9cstabilizerxe2x80x9d oligomer, as provided for in this invention, is unique in that unlike prior base-stacking inventions, it functionally serves two purposes (i.e., to hinder reannealing of complementary amplicons during electronic addressing of the biotinylated target amplicons, and to provide a base-stacking energy moiety for interaction with the second oligomer. This combined functionality effectively lessens the complexity of SNP determination in a microchip format).
Application of site-specific electronic biasing can allow for directed influencing of the ionic environment at the site of hybridization as well as continuous adjustment of hybridization conditions both during and after hybridization. Such manipulation of electronic environment (specifically the dielectric constant of the solution) can be used to influence directly the base-stacking energies between oligonucleotide probes. Additionally, hybridization is greatly accelerated by the concentration achieved during local electronic addressing. Such a system is also highly flexible in that it allows one to take advantage of both thermal and/or electronic discrimination after hybridization. Moreover, electronic biasing equally facilitates distinguishing hybridization mismatches occurring at the terminal nucleic acid pairs of a hybridized duplex as well as destabilizing mismatches occurring internally (e.g., due to destabilizing caused by misalignment of the base pairs). This ability to detect mismatches allows the current invention to be less restricted in choices for positioning the location of SNP bases on probes although generally, for purposes of this invention, mismatches are desired to occur at the terminal base of a probe. For instance, the SNP relevant base may be incorporated as the terminal base of the reporter probe such that when the stabilizer and reporter probes are annealed to the amplicon, the SNP relevant base will lie adjacent to one of the terminal bases of the stabilizer when both the stabilizer and reporter are annealed adjacently to one another on a target nucleic acid strand.
Sensitivity and robustness may further be enhanced by the additional inclusion of yet another probe (i.e., the xe2x80x9cinterferingxe2x80x9d probe) designed to be complementary to the non-labeled strand of the amplicon. Use of this probe further helps to compete away the undesired non-labeled amplicon strand from reannealing to the labeled strand.
In another format of this system, when the stabilizer probe is anchored (i.e. xe2x80x9ccapture downxe2x80x9d format), the system is also simple and multiple amplicons may be placed at the same capture site. These may then be probed simultaneously or sequentially. Generally, although not exclusively, the stabilizer probe will be anchored to the substrate at its 5xe2x80x2 end. Such an arrangement necessarily provides that the SNP base will be complementary to either the 3xe2x80x2 base of the stabilizer/capture or the 5xe2x80x2 base of the reporter probe. Conversely, if the 3xe2x80x2 end of the stabilizer/capture is anchored, then the SNP base will be complementary to either the 5xe2x80x2 base of the stabilizer/capture or the 3xe2x80x2 base of the reporter probe.
By way of example, in implementation of this capture down format, a target nucleic acid is first amplified, such as by PCR or SDA. The amplified dsDNA product is then denatured and addressed to a specified capture site on the microchip surface that has an anchored stabilizer/capture moiety. In a preferred embodiment, the complementary strand to the desired amplification product strand is kept from reannealing to the desired strand by the stabilizer/capture oligomer that, as described above, serves as a first probe that also participates in base-stacking with a second reporter probe. As in the amplicon down method, the stabilizer/capture oligomer as provided for in this invention is unique in that unlike prior base-stacking inventions, it functionally serves two purposes (i.e., to hinder reannealing of complementary amplicons during electronic addressing of the target amplicons and to provide a base-stacking energy moiety for interaction with reporter oligomer thereby lessening the complexity of SNP determination in a microchip format). As with the target down format, interfering probes may be used. Moreover, multiple amplicons may be probed at any particular capture site.
In yet another format, multiple SNPs in a target sequence may be detected. In this format, either of the above mentioned amplicon down or capture down formats may be employed. In this format, multiple base-stacking may be used to resolve the presence of closely spaced SNPs at a single locus. For example, where two SNPs are closely spaced, at least two short reporter oligonucleotides may be base-stacked against a longer stabilizer oligonucleotide. Each reporter may be labeled with a different fluorophore specific for the allele that occurs at each site. For instance, if a locus has two SNPs in close proximity to one another, reporter probes incorporating the wild-type and mutant bases of each SNP site, each containing a different fluorophore may be used to determine which allele is present.
In yet another embodiment of the invention, SNPs in a target nucleic acid are determined using combined base-stacking energies derived from both 5xe2x80x2 and 3xe2x80x2 ends of a single reporter probe. In this embodiment, the target nucleic acid is amplified (such as by PCR and preferably via the strand displacement amplification (SDA) technique) such that two spaced amplicons of the target are generated. The two amplicons (a first and a second amplicon) may be from the same genetic locus wherein the sequences are closely spaced, or may be from divergent or unrelated genetic loci. In either case, both the amplicon down and the capture down formats may be used. In the case where the capture down format is used, the stabilizer/capture is designed as a xe2x80x9cbridgingxe2x80x9d stabilizer/capture probe to capture both amplicons in a spaced apart fashion so that at least one reporter probe, which may or may not contain SNP sequence at one or the other end, can be xe2x80x9cnestedxe2x80x9d between the amplicons. Where the amplicon down format is used, only one of the amplicons is anchored and a xe2x80x9cbridgingxe2x80x9d stabilizer/capture probe having sequence complementary to the anchored amplicon and the non-anchored amplicon is employed to hybridize the amplicons in a spaced apart fashion allowing at least one reporter probe to be nested. Where multiple SNPs are associated at such a loci, more than one SNP containing reporter probe may be nested and take advantage of multiple base-stacking energies.
In the case where the amplicons are from different loci, the amplicons may be brought into close proximity with one another using either an anchored bridging stabilizer/capture probe, or an anchored amplicon and a bridging stabilizer/capture probe as described above. The presence of both amplicon sequences may be detected using a reporter probe designed to nest between the captured amplicons using base-stacking energies to stabilize the reporter hybridization as described above. As with the earlier described formats, the reporter probe may incorporate at either and/or both its 5xe2x80x2 and 3xe2x80x2 ends SNP or wild-type sequence associated with either or both loci.
In a further embodiment, the SNP containing region may contain multiple SNPs and reporter probes can be designed so that more than one reporter probe is used to nest between the first and second amplicons such that each reporter has at least one nucleic acid base on either its 3xe2x80x2 or 5xe2x80x2 end corresponding to a SNP. Thus, such a system can benefit from both multiple reporter signals and multiple base-stacking energies from nesting probes that possess either a single base corresponding to either SNP or wild-type at either the 3xe2x80x2 or 5xe2x80x2 end, or that contain such bases at both 3xe2x80x2 and 5xe2x80x2 ends, thereby increasing sensitivity.
In another embodiment the stabilizer oligomers are generally 20 to 44-mers and preferably about 30-mers, while the reporter probes are generally 10 to 12-mers and preferably about 11-mers. The lengths of such probes are highly effective in accordance with their use in an electronically addressable microchip format. Reporter probes shorter than 8-mers are generally not functional in the ionic environment of the current system.
In the preferred embodiment of the invention, electronically aided hybridization is utilized in the process. In one aspect, during the hybridization of the nucleic acid target with the stabilizer probe and/or the reporter probe, electronic stringent conditions may be utilized, preferably along with other stringency affecting conditions, to aid in the hybridization. This technique is particularly advantageous to reduce or eliminate slippage hybridization among probes and target, and to promote more effective hybridization. In yet another aspect, electronic stringency conditions may be varied during the hybridization complex stability determination so as to more accurately or quickly determine whether a SNP is present in the target sequence.
Hybridization stability may be influenced by numerous factors, including thermoregulation, chemical regulation, as well as electronic stringency control, either alone or in combination with the other listed factors. Through the use of electronic stringency conditions, in either or both of the target hybridization step or the reporter oligonucleotide stringency step, rapid completion of the process may be achieved. Electronic stringency hybridization of the target is one distinctive aspect of this method since it is amenable with double stranded DNA and results in rapid and precise hybridization of the target to the capture site. This is desirable to achieve properly indexed hybridization of the target DNA to attain the maximum number of molecules at a test site with an accurate hybridization complex. By way of example, with the use of electronic stringency, the initial hybridization step may be completed in ten minutes or less, more preferably five minutes or less, and most preferably two minutes or less. Overall, the analytical process may be completed in less than half an hour.
As to detection of the hybridization complex, it is preferred that the complex is labeled. Typically, in the step of determining hybridization of probe to target, there is a detection of the amount of labeled hybridization complex at the test site or a portion thereof. Any mode or modality of detection consistent with the purpose and functionality of the invention may be utilized, such as optical imaging, electronic imaging, use of charge-coupled devices or other methods of quantification. Labeling may be of the target, capture, or reporter. Various labeling may be by fluorescent labeling, colormetric labeling or chemiluminescent labeling. In yet another implementation, detection may be via energy transfer between molecules in the hybridization complex. In yet another aspect, the detection may be via fluorescence perturbation analysis. In another aspect the detection may be via conductivity differences between concordant and discordant sites.
In yet another aspect, detection can be carried out using mass spectrometry. In such method, no fluorescent label is necessary. Rather detection is obtained by extremely high levels of mass resolution achieved by direct measurement, for example, by time of flight or by electron spray ionization (ESI). Where mass spectrometry is contemplated, reporter probes having a nucleic acid sequence of 50 bases or less are preferred.
It is yet a further object of this invention to provide methods that may effectively provide for genetic identification.
It is yet a further object of this invention to provide systems and methods for the accurate detection of diseased states, especially clonal tumor disease states, neurological disorders and predisposition to genetic disease.
It is yet a further object of this invention to provide a rapid and effective system and methods for identification, such as in forensics and paternity applications.
Yet a further object of the invention is to identify SNPs in infectious organisms such as those responsible for antibiotic resistance or that can be used for identification of specific organisms.